Light microscopes, transmission electron microscopes, scanning electron microscopes and other instruments are extensively used to understand the ultrastructure of a wide variety of synthetic and biological materials in numerous areas of science and technology. For example, light microscopy samples are used for research to identify the development of different organs in animals and plants. In addition, one major use of LM samples is in the histopathologic examination of biopsy samples of tissues suspected of disease. TEM is used to study both biological samples as well as non-biological samples and can even provide atomic scale resolution. TEM is routinely used to investigate metal grain structures, the micro and nano-structures of polymers, semiconductor devices such as computer chips, and to visualize the organelle and molecular organization of cells. Such images are capable of resolution down to approximately 0.1 nm, although this is usually not quite possible with biological materials. TEM and LM specimens are commonly sliced or otherwise prepared into thin cross-sections to enable electrons or photons, respectively, to traverse through the specimen to create an image. SEM is similar to TEMs in that it uses electrons to create an image of the target/sample. However, the resolution of the SEM is typically not as fine as that of the TEM, yet high resolution versions are capable of molecular level resolution of approximately 5 nm. Due to the SEM's ability to image the surface aspect of bulk materials, specimen preparation does not usually entail slicing the specimen into cross-sections.
Study objects for microscopy are prepared in multiple ways depending upon the type of material to be examined, and the type of microscopy to be used. Biological materials require special handling to preserve the structure of the material when it will be examined in the electron microscope, and secondarily to enhance or enable imaging.
Both SEM and TEM instruments perform their imaging in a vacuum (the absence of air or other gases). Since biological materials are generally 50 to 95% water, if these were placed directly within the vacuum the water would evaporate and the specimen would collapse. Consequently both SEM and TEM samples have the water removed after the structure is strengthened with chemicals such as glutaraldehyde, formaldehyde, and osmium tetroxide.
TEM samples must be very thin (typically about 40-100 nm) in order for the electrons used for imaging to be “transmitted” or passed through the sample. To cut specimens into such thin sections the water in the specimen is replaced with plastic that is hardened in place. This plastic supports the sample as it is sliced very thin using a device called an ultra-microtome.
LM specimens, especially those of biological origin, are generally also sectioned in order to provide cross-sections for viewing, and to allow photons (light) to be transmitted through the specimen. As with TEM, LM sections are also embedded to support the specimen during sectioning, however, different generally softer plastics or wax are used as the embedding materials. Water that is frozen with additional materials to enable the ice to be softer, provide better support of the tissue, and reduce ice crystal damage during freezing can also be used.
Because of the delicate nature of microscopy samples and the resolution and power of LM, SEM and TEM analysis, sample preparation requires highly skilled and delicate manipulation. For example, the preparation of LM, SEM and TEM samples may require from between 20 to 40 fluid exchanges. Such samples may be prepared in large quantity and their quality, analysis and identification may have significant downstream impact, such as for biopsy samples, for example. Thus, methods and devices for their preparation have been devised that are either highly cumbersome and/or inadequate for their needs.
For example, U.S. Pat. No. 5,080,869 to McCormick discloses an apparatus and method for preparing samples for histological examination. The device comprises a cassette suitable for holding a sample with perforation in the wall and floor to drain fluid. A stack of such cassettes can be put in a container for the process of fixation. As illustrated by McCormick, inside the container and the cassette are large amounts of dead space resulting in the need for large volumes of fixation media, rinses, and other solutions in order to adequately process the sample. Since many of the chemicals used are noxious and some are expensive to purchase or dispose of, large dead-space volumes are not desirable. In addition, such cassettes are not intended for the preparation and handling of the circa 1 mm specimens typical in biological TEM and many clinical histopathology or biopsy specimens and hence such small specimens can readily become lost or misplaced.
U.S. Pat. No. 5,543,114 to Dudek discloses a unitary biological specimen processing apparatus. The apparatus comprises a container having a lid with apertures in it for straining fluid from the container. As disclosed by Dudek, a sample is placed in the container and fixation fluids entered in one end and emptied out the other end. As can be seen in the Dudek figures, the container has a large volume of dead space in which the sample can be lost or damaged. Further, the sample can not be visually inspected nor can the sample be sectioned or stored in the container.
Similarly, U.S. Pat. No. 7,179,424 to Williamson et al. discloses a cassette for handling and holding tissue samples during processing. As with other, similar devices, the cassette disclosed by Williamson includes a large volume of dead space, and provides little ability to visually inspect the samples within. Further, the cassettes taught by Williamson are not amendable to simultaneous use with other cassettes thus limiting their ability for high-throughput use. In addition, as taught by Williamson et al., the cassettes are complicated devices having relatively high costs and not allowing for sectioning of the sample held within.
Further, most other imaging and analytical methods used to analyze specimens on such small scales could benefit from methods and/or devices that facilitate and standardize the process of preparing such samples. Such applications require the handling, processing, and identification of small specimens for analyses such as, for example, secondary ion mass spectroscopy (SIMS), electron spectroscopy for chemical analysis (ESCA) which is also known as x-ray photoelectron spectroscopy (XPS) and atom probe tomography. Matrix-assisted laser desorption ionization (MALDI), and many other analytical and imaging instrument preparation procedures, are also within the scope of this invention.
Thus, the need exists for a low-cost device and method that allows for the parallel uses of fixation and subsequent processing of multiple samples, providing for a high-throughput microscopy specimen preparation system that further allows for the tracking and identification of samples held both on a short-term scale and for long-term storage.
Therefore, there is a need for devices and methods to be used in the preparation of TEM grids and specimens for TEM, SEM, LM, SIMS, ESCA, XPS and MALDI that provide for easier preparation of the grids and specimens for analysis and that may allow for more efficient storage and handling.